Isotopic labeling-assisted metabolomics using LC-MS

Abstract

Metabolomics has emerged as the latest of the so-called "omics" disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC-HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC-MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC-MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.

Fig. 1

Different strategies for using SIL to assist metabolomics studies. a ¹³C, ¹⁵N, and ³⁴S-enriched substances are not chromatographically separated from the corresponding natural isotopologues, thus the non-labeled and the labeled isotopologues elute at the same retention time with identical peak profiles. b For non-targeted annotation of an organism’s metabolome the organism can be cultivated in parallel using differently isotopologue-enriched nutrition sources (e.g., ¹²C and ¹³C glucose as sole carbon source). The extracts are subsequently mixed and measured with LC–HRMS. The resulting data pattern helps in the extraction of true biological signals. c Absolute compound quantification using an authentic, labeled standard or relative quantification using a stock of globally labeled sample extract of the same organism for inter-experiment comparison. d Metabolism experiment using natural and fully labeled tracer substances enables metabolism studies and greatly helps to separate products of metabolism from other biological signals. In contrast with metabolism studies, fluxomics experiments only spike with the labeled tracer. e Derivatisation using non-labeled and labeled derivatisation agents enables rapid recovery of many metabolites belonging to the same chemical groups (e.g., alcohols, acids …)

Fig. 2

3D Plots obtained from F. graminearum. a Unprocessed LC–HRMS full-scan chromatograms of a mixture of supernatants from cultivation of F. graminearum on both ¹²C and ¹³C glucose. The circle marks an ion pair (the ¹²C and the corresponding ¹³C-labeled monoisotopic ions) originating from the same metabolite. After processing the spectrum with MetExtract, only ions having a labeled pendant are not removed (b). Thus, only the non-labeled ions remain in the circled area. (3D view generated with Ref. [37])